Bacillus thuringiensis ( "B.t.") is a gram-positive soil bacterium that produces proteinaceous crystalline inclusions during sporulation. These B.t. crystal proteins are often highly toxic to specific insects. Insecticidal activities have been identified for crystal proteins from various B.t. strains against insect larvae from the insect orders Lepidoptera (caterpillars), Diptera (mosquitos, flies) and Coleoptera (beetles).
Recently, certain B.t. strains and B.t. crystal proteins have been reported as having activity against non-insect species such as nematodes. The term "insecticidal," as used herein with reference to B.t. strains and their crystal proteins, is intended to include such pathogenic activities against non-insect species.
Individual B.t. crystal proteins, also called delta-endotoxins or parasporal crystals or toxin proteins, can differ extensively in their structure and insecticidal activity. These insecticidal proteins are encoded by genes typically located on large plasmids, greater than 30 megadaltons (mDa) in size, that are found in B.t. strains. A number of these B.t. toxin genes have been cloned and the insecticidal crystal protein products characterized for their specific insecticidal properties. A good review of cloned B.t. toxin genes and crystal proteins is given by Hofte et al., Microbiol. Rev. 53:242-255 (1989), who also propose a useful nomenclature and classification scheme that has been adopted here.
The insecticidal properties of B.t. have been long recognized, and B.t. strains have been incorporated in commercial biological insecticide products for over thirty years. Commercial B.t. insecticide formulations typically contain dried B.t. fermentation cultures whose crystal protein is toxic to various insect species.
Traditional commercial B.t. bioinsecticide products are derived from "wild-type" B.t. strains, i.e., purified cultures of B.t. strains isolated from natural sources. Newer commercial B.t. bioinsecticide products are based on genetically altered B.t. strains, such as the transconjugant B.t. strains described in U.S. Pat. No. 5,080,897 issued to Gonzalez, Jr., et al. on Jan. 14, 1992, and in U.S. Pat. No. 4,935,353 issued to Burges et al. on Jun. 19, 1990.
Recombinant B.t. strains, with novel complements of B.t. toxin genes providing wide spectrum and/or enhanced insecticidal activity, are likely to be incorporated into commercial B.t. bioinsecticides in the foreseeable future.
All B.t.-based bioinsecticides have the advantage of being selectively toxic to specific target insect species without exhibiting any toxicity towards vertebrates. A drawback to B.t. bioinsecticides is that the persistence of insecticidal activity is limited. The insecticidal half-life of B.t. bioinsecticides is typically less than one week in duration, even with protective adjuvants that are typically employed in commercial B.t. formulations.
Studies on loss of insecticidal activity in B.t. proteins have identified solar irradiation as a cause and have recommended the use of photoprotectants in B.t. formulations; see, for example, Pozsgay et al., J. Invertebr. Pathol. 50:246-253 (1987) and Morris, Canadian Entomol. 115:1215-1227 (1983): Cell encapsulation and cell treatment technologies such as described in U.S. Pat. Nos. 4,695,455 and 4,695,462, both issued to Barnes et al. on Sep. 22, 1987, have also been employed to protect the insecticidal toxin activity of the B.t. protein against environmental factors.
It has long been recognized that the insecticidal activity of B.t. proteins is induced, at least in part, by the action of proteolytic enzymes on B.t. protein which has been ingested by a susceptible insect species. Hofte et al., Microbiol. Rev. 53:242-255 (1989), note that B.t. crystalline protein dissolves in the larval insect midgut and that many of these B.t. proteins are protoxins that are proteolytically converted into smaller toxic polypeptides, i.e., activated toxin, in the insect midgut.
Researchers have reported the existence of proteases in B.t., but the role of these proteases in the insecticidal activity of B.t. proteins or, more generally, in the physiology of B.t. is unclear because of the contradictory results reported.
The role of B.t. proteases in B.t. protein solubilization is described by Chestukhina et al., Biokhimiya 43:857-864 (1978), who carried out dissolution studies with B.t. crystalline proteins and concluded that degradation of B.t. proteins during dissolution was facilitated by the presence of serine proteases, metalloproteases and leucine antipeptidase in the B.t. crystal protein. Similarly, Thurley et al., FEMS Microbiol. Lett. 27:221-225 (1985), reported that a cystein-like crystal-associated protease assisted in the solubilization of a B.t. crystal protein.
A role of B.t. proteases in insecticidal activity is described by Chilcott et al., FEMS Microbiol. Lett. 18:37-41 (1983), who reported that two different proteolytic activities were associated with B.t. crystal protein and that B.t. with reduced proteolytic activity exhibited lower insect toxicity. Bulla, Jr. et al., J. Bacteriol. 130:375-383 (1977), likewise concluded that B.t. crystal proteins have an autolytic activity which apparently involves a sulfhydryl protease that is activated under alkaline conditions (such as found in an insect midgut) and that renders the B.t. protein insecticidal.
An opposite conclusion was reached by Pfannensteil et al., FEMS Microbiol. Lett. 21:39-42 (1984), who reported that B.t. crystal protein treated to reduce the crystal-associated protease activity exhibited no difference in insecticidal activity from the untreated crystal protein (which was from the same B.t. subspecies as that of Chilcott et al.). A similar conclusion was reached by Bibilos et al., Canad. J. Microbiol. 34:740-747 (1988) who described the role of B.t. proteases, primarily neutral metalloproteases, in effecting activation of B.t. protein protoxin to active toxin, but concluded that this is unnecessary, since the same result is accomplished in the insect midgut, presumably by insect-derived proteases.
In contrast to the results reported by Chilcott et al. and Pfannensteil et al., Pearson et al., J. Appl. Bacteriol. 65:195-202 (1988), proposed a negative role for B.t. proteases in the insecticidal activity of B.t. crystal protein. Pearson et al. concluded that more than one type of protease was present in B.t. crystal protein produced by sporulated, lysed B.t. cells and that a decline of insecticidal activity might be associated with proteolytic action.
Some B.t. proteases have been identified, at least in part, in the literature. Li et al., Applied Microbiol. 39:354-361 (1975), describe a partially purified extracellular metalloprotease from B.t. having a molecular mass of about 38 kilodaltons (kDa). Lecadet et al., Eur. J. Biochem. 79:329-338 (1977), describe an extracellular serine protease from B.t. having a molecular mass of about 23 kDa. Stepanov et al., Biochem. Biophys. Res. Comm. 100:1680-1687 (1981), disclose the N-terminal amino acid sequence of a purified extracellular serine protease from B.t. Kunitate et al. , Agric. Biol. Chem. 53:3251-3256 (1989), describe a purified SH-containing serine protease from B.t. whose N-terminal sequence was similar to that reported by Stepanov et al. for their purified protease.
An isolated, cloned B.t. protease gene is described by Lovgren et al., Molecul. Microbiol. 4:2137-2146 (1990), who report the nucleotide sequence of a B.t. derived gene that encodes a neutral metalloprotease, which was found to be toxic when injected into a lepidopteran insect.
The nucleotide sequence of the promoter region of a B.t. protease gene is disclosed by Geiser in PCT International Patent Publication No. WO 92/14826 dated Sep. 3, 1992, of Ciba-Geigy AG, where the promoter is operatively linked with a heterologous gene, such as .beta.-galactosidase.
Although the published literature on the cloning and characterization of B.t.-derived protease genes is limited, much work has been carried out on identifying proteases and protease genes in other Bacillus species, particularly Bacillus subtilis: see Pero et al., "Proteases" in Bacillus subtilis and other Gram-Positive Bacteria, Amer. Soc. Microbiol. , Washington, D.C., 1993, pages 939-952. The cloning of the subtilisin gene of B. subtilis, which encodes an alkaline protease (also called subtilisin or serine protease), is reported by Stahl et al. , J. Bacteriol. 158:411-418 (1984). Another B. subtilis protease gene, one that encodes a neutral metalloprotease, was cloned by Yang et al., J. Bacteriol. 160:15-21 (1984), who used a deleted version of the neutral protease gene to displace the wild-type neutral protease gene in B. subtilis. Proteolytic activity in the mutant was only 20% of the wild-type strain, and another mutant, deleted in both the alkaline protease gene and the neutral protease gene, produced no detectable levels of proteolytic activity. A major extracellular protease of Bacillus cereus was purified by Sidler et al., Biol. Chem. Hoppe-Seyler 367:643-657 (1986), who reported the N-terminal amino acid sequence to be 45% homologous to that of the B. subtilis neutral protease.
U.S. Pat. No. 4,828,994, issued to Fahnestock et al. on May 9, 1989, discloses production of genetically altered strains of Bacillus subtilis, which is rendered incapable of synthesizing the proteolytic enzyme subtilisin by replacing the native chromosomal DNA comprising the subtilisin gene with a DNA sequence comprising a subtilisin gene which has an inactivating DNA sequence inserted therein. The purpose is to insert into the subtilisin gene a functional gene coding for a protein which confers a phenotypic trait, such as resistance to a selected antibiotic to facilitate identification of the altered microorganism and subsequent transfer of the inactivated gene into other bacterial strains. "Subtilisin" as used by Fahnestock et al. refers to the enzyme alkaline serine protease, without regard to the species of Bacillus in which it is produced.
U.S. Pat. No. 4,766,077, issued to Orser et al. on Aug. 23, 1988, relates to a method for modifying ice nucleation bacteria in vitro to confer an ice nucleation deficient phenotype. Modification is accomplished by deletion, substitution, insertion, inversion or transversion of a DNA segment within the gene locus responsible for the ice nucleation phenotype. The mutations are limited to the particular gene locus so that the modified microorganisms are genetically stable and free from random mutations which might adversely affect their competitive fitness. The modified microorganisms are useful for prevention of frost damage to susceptible plant hosts.